Terutada Kobayashi

Orthotopic implantation mouse model and cDNA microarray analysis indicates several genes potentially involved in lymph node metastasis of colorectal cancer

In colorectal cancer (CRC) patients, metastasis to the regional lymph node (LN) is an important first step in the dissemination of cancers. To identify the genes possibly involved in LN metastasis of CRC, we analyzed LN metastases in an orthotopic implantation mouse model with 22 CRC cell lines using Matrigel, an extracellular matrix protein derived from mice sarcoma, and combined the data with gene expression profiles of cDNA microarray of those cell lines. With this implantation analysis, the incidence of LN metastasis was 60% in 228 orthotopically implanted mice and varied from 100% to 0% among the cell lines. KM12c and Clone A showed LN metastasis in all orthotopically implanted mice, but DLD‐1, HCT‐8, and SW948 did not show LN metastases at all. In contrast, the incidence of liver and lung metastasis in 22 CRC cell lines was 13% and 1%, respectively. Combining those data with cDNA microarray in vitro, we isolated 636 genes that were differentially expressed depending on the incidence of LN metastasis. Among those genes, the expression level of ring finger protein 125 (RNF125), previously known as an E3 ubiquitin ligase in T cell activation, was significantly different between primary tumors in Stage III CRC patients with LN metastasis and Stage II patients without LN metastasis. In conclusion, the orthotopic implantation mice model with Matrigel was useful, and we isolated candidate genes such as RNF125 that possibly play an important role in LN metastasis of CRC cells. (Cancer Sci 2008; 99: 711–719)

Cancer-associated splicing variants of the CDCA1 and MSMB genes expressed in cancer cell lines and surgically resected gastric cancer tissues

BACKGROUND Alternative splicing is a molecular mechanism by which different combinations of exons can be alternatively spliced to produce different mRNA isoforms. Recently, several databases have been published to predict the alternative splicing of mRNA; cancer-specific alternative splicing has also been predicted with these databases. Those variants may be potentially useful targets for cancer therapy, however, the accuracy and veracity of these databases have yet to be confirmed. METHODS In this study, we analyzed 17 genes by reverse transcriptase-polymerase chain reaction (RT-PCR) that were predicted to have cancer-specific alternative splicing by using the splicing database, the Alternative Splicing Annotation Project (ASAP) by Lee et al, between 38 cancer cell lines from various organs and 9 corresponding normal tissues. By designing 2 types of primer sets for RT-PCR including (1) primers flanking the alternatively spliced exons and (2) primers spanning the exon/exon junctions, cancer-associated splicing variants were investigated. RESULTS The alternatively splicing events were detected in 15 of 17 genes (88%); 35 of 43 variants (81%) were detected successfully with RT-PCR. Among these variants, M-RIP, HYAL2, CDCA1, and MSMB genes showed differential expressions between cancer cell lines and corresponding normal tissues. Furthermore, RT-PCR with surgically resected gastric cancer tissues (diffuse type, 6; intestinal type, 4) confirmed that 2 variants of CDCA1 were upregulated in cancer tissues, whereas both variants of MSMB were expressed predominantly in normal tissues. CONCLUSION Alternative splicing variants, especially in CDCA1, were detected in this study and may be potentially useful as diagnostic markers and/or novel targets for anticancer therapy.

Cronkhite-Canada syndrome complicated with huge intramucosal gastric cancer

Cronkhite-Canada syndrome (CCS) is a rare nonhereditary disorder with gastrointestinal polyposis and associated ectodermal changes. This report documents a 59-year-old Japanese man with CCS who underwent a total gastrectomy for gastric tumors. The resected specimen showed a huge gastric adenocarcinoma with numerous polyps throughout the stomach. The cancer was pathologically limited to within the mucosa and its histological structure resembled that of hyperplasia in CCS polyps, which led us to suppose that the carcinoma had arisen from hyperplastic CCS polyps. These results urged us to study the phenotypic expression of mucins, which revealed MUC2(−) and MUC5AC(+) and supported the diagnosis of the tumor as a gastric-type well-differentiate adenocarcinoma. A literature search revealed that 32 gastric carcinomas which developed in patients with CCS were mostly limited to within the submucosa in spite of their huge sizes, and such cancer development in CCS polyposis is therefore not considered to be unusual.

Interleukin-12 administration is more effective for preventing metastasis than for inhibiting primary established tumors in a murine model of spontaneous hepatic metastasis

AbstractPurpose. The effect of interleukin-12 (IL-12) on tumor growth at the primary site and its therapeutic efficacy against metastasis were examined using a model of spontaneous hepatic metastasis. Methods. IL-12 was peritumorally injected into RL♂F1 tumor-bearing BALB/c male mice at the different tumor stage. Results. Striking inhibition of hepatic metastasis in both athymic and euthymic mice was induced by the administration of IL-12 irrespective of the stage of tumor progression. In contrast, IL-12 failed to produce any antitumor effect in athymic mice which lack conventional T cells. These results suggest that the antitumor effect of IL-12 is mediated mainly by T cells, and that the antimetastatic effect is mediated mainly by natural killer (NK) and/or NKT cells. Next we examined the direct effect of IL-12 on these cells. IL-12-induced T-cell proliferation was remarkably augmented in the early stage, then decreased dramatically in the advanced stage, while IL-12-induced cytotoxic activity, mediated by NK and NKT cells, was not attenuated even in the advanced stage. This dissociation in IL-12 responsiveness appeared to be the reason for the remarkable antimetastatic effect but insufficient antitumor effect of IL-12 in the advanced stage. Conclusion. The findings of this study support the clinical use of IL-12 for immunotherapy against either occult or evident liver metastasis.

Giant T4 rectal carcinoma mimicking urinary bladder adenocarcinoma accurately diagnosed by immunohistochemistry and successfully treated with total pelvic exenteration: report of a case.

A 54-year-old man, with the chief complaints of hematuria, pollakisuria, and pneumaturia, was referred to our hospital, with a diagnosis of giant urinary bladder adenocarcinoma with massive invasion to the rectum. On the basis of the radiological diagnosis and findings of hematoxylin and eosin (H&E) of biopsy specimens, it was difficult to conclude whether the adenocarcinoma originated in the bladder or in the rectum. The immunohistochemical staining of the biopsy specimens showed cytokeratin 7 (CK7)(−) and CK20(+), which supported the notion that the adenocarcinoma possibly originated from the rectum. Although the prognosis of T4 bladder adenocarcinomas has been reported to be quite poor in comparison with that of transitional cell carcinomas, the postoperative prognosis of T4 rectal adenocarcinomas has been reported to be more favorable and such tumors are recommended to be surgically resected. Because no distant metastasis was detected, the patient underwent total pelvic exenteration with a reconstruction of the ileal conduit. Although the resected tumor measured 12 cm in diameter with n1 metastasis, the radial margin was cancer-negative, and the tumor was curatively resected. The immunohistochemical diagnosis of the resected tumor showed carcinoembryonic antigen(+), CK7(−), CK20(+), thrombomodulin(−), and uroplakin(−), which supported the rectal origin. At present the patient is undergoing postoperative adjuvant chemotherapy for rectal cancer.